In recent years, the manufacture and commercialization of complex carbohydrates, particularly secreted oligosaccharides, have increased significantly due to their roles in numerous biological processes occurring in living organisms. Secreted oligosaccharides such as human milk oligosaccharides (HMOs), mucin oligosaccharides and Lewis type oligosaccharides have gained much interest and have become important commercial targets for nutrition and therapeutic applications. In particular, the synthesis of these oligosaccharides has increased significantly due to their role in numerous biological processes occurring in humans.
Many human milk oligosaccharides contain sialosides, mainly N-acetyl-neuraminic acid, that is most frequently found in the terminal end of oligosaccharides. The linkages of N-acetyl-neuraminic acid in which it is bound to galactose and N-acetyl-glucosamine are α-2,3- and α-2,6-ketosidic bonds. This terminally exposed position allows sialoconjugates to be recognized by receptors of cells, viruses and bacteria, thus to be involved in a wide variety of biological processes.
The pentasaccharide 6′″-O-sialyl-LNT (LST b, Galβ(1-3)-[Neu5Acα(2-6)-]GlcNAcβ(1-3)-Galβ(1-4)-Glc) is a human milk oligosaccharide and represents a common structural element in some other sialylated human milk oligosaccharides (Urashima et al.: Milk Oligosaccharides, Nova Medical Books, NY, 2011); they are listed in Table 1.
TABLE 1Selected sialylated HMOsNo HMO nameHMO structure1LST bGalβ(1-3)-[Neu5Acα(2-6)-]GlcNAcβ(1-3)-Galβ(1-4)-Glc2F-LST bFucα(1-2)-Galβ(1-3)-[Neu5Acα(2-6)-]GlcNAcβ(1-3)-Galβ(1-4)-Glc3DS-LNTNeu5Acα(2-3)-Galβ(1-3)-[Neu5Acα(2-6)-]GlcNAcβ(1-3)-Galβ(1-4)-Glc4FDS-LNT I Neu5Acα(2-3)-Galβ(1-3)-[Neu5Acα(2-6)-]{Fucα(1-4)-}GlcNAcβ(1-3)-Galβ(1-4)-Glc5FDS-LNT IINeu5Acα(2-3)-Galβ(1-3)-[Neu5Acα(2-6)-]GlcNAcβ(1-3)-Galβ(1-4)-[Fucα(1-3)-]Glc6FS-LNH INeu5Acα(2-3)-Galβ(1-3)-[Neu5Acα(2-6)-]GlcNAcβ(1-3)-[Galβ(1-4)-{Fucα(1-3)-}GlcNAcβ(1-6)-]Galβ(1-4)-Glc7DS-LNH II Neu5Acα(2-3)-Galβ(1-3)-[Neu5Acα(2-6)-]GlcNAcβ(1-3)-[Galβ(1-4)-GlcNAcβ(1-6)-]Galβ(1-4)-Glc8FDS-LNH INeu5Acα(2-3)-Galβ(1-3)-[Neu5Acα(2-6)-]GlcNAcβ(1-3)-[Fucα(1-2)-Galβ(1-4)-GlcNAcβ(1-6)-]Galβ(1-4)-Glc9FDS-LNH IINeu5Acα(2-3)-Galβ(1-3)-[Neu5Acα(2-6)-]GlcNAcβ(1-3)-[Galβ(1-4)-{Fucα(1-3)-}GlcNAcβ(1-6)-]Galβ(1-4)-Glc10 TS-LNHNeu5Acα(2-3)-Galβ(1-3)-[Neu5Acα(2-6)-]GlcNAcβ(1-3)-[Neu5Acα(2-6)-Galβ(1-4)-GlcNAcβ(1-6)-]Galβ(1-4)-Glc
DS-LNT has recently been found to be preventive against necrotising enterocolitis (NEC) in rat model (Jantscher-Krenn et al. Gut 61, 1417 (2012), WO 2012/106665).
More thorough research studies of cell-cell interactions, mechanism of cancer malignancy and/or determination of the bioactive conformation of compounds that are involved in protein-glycan recognition processes have required greater quantities of such oligosaccharides, but present enzymatic and/or microbial methods have not been able to provide with them in sufficient amounts and/or purities. Synthetic chemical methods, adapted to provide them in sufficient purity, have required many expensive reaction steps and the extensive use of protection-deprotection sequences during the synthesis, and afford compounds only in mg quantities, as demonstrated by the synthesis of an α(2-3)/α(2-6)-disialyl lactotetraosyl ceramide and a disialyl Lewis A ganglioside (Ando et al. J. Carbohydr. Chem. 20, 425 (2001), Carbohydr. Res. 338, 503 (2003)).
There has been a need, therefore, for an efficient method of making a diverse group of sialylated and/or fucosylated LNT derivatives and their analogs. There has been a particular need for a method that can be readily scaled-up for potential industrial use.